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Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs).

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Bottom Line: The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk.The method is rapid and the results are reproducible.HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

Background: Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.

Results: An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.

Conclusion: This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

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Multilocus allele analysis of Brucella species and biovar type strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella species and biovars. The allele number reflects the total number of repeats calculated for each locus. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. The star next to the VNTR Locus-4 allele in some samples shows a match with the alternative allele ladder indicating an 11-bp insertion in the non-repeat region of the amplicon.
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Figure 4: Multilocus allele analysis of Brucella species and biovar type strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella species and biovars. The allele number reflects the total number of repeats calculated for each locus. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. The star next to the VNTR Locus-4 allele in some samples shows a match with the alternative allele ladder indicating an 11-bp insertion in the non-repeat region of the amplicon.

Mentions: A panel of 19 pure Brucella cultures, composed of all of the Brucella species and biovar FAO/WHO Reference Strains, was assayed by the HOOF-Print procedure. The amplicons obtained from each locus were sized and the numbers of repeat units were determined. The data were compiled in chart format for easy comparison (Fig. 4). The collective alleles amplified from a given isolate formed its genetic fingerprint. Each fingerprint was unique and each of the type strains could be easily differentiated. However, parts of the fingerprint patterns were similar among the related biovars of a single species. The VNTR Locus-8 locus, for example, contained two repeats in all B. abortus biovars. Similarly, the VNTR Locus-3 locus contained a single repeat in the three B. melitensis biovars.


Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs).

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Multilocus allele analysis of Brucella species and biovar type strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella species and biovars. The allele number reflects the total number of repeats calculated for each locus. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. The star next to the VNTR Locus-4 allele in some samples shows a match with the alternative allele ladder indicating an 11-bp insertion in the non-repeat region of the amplicon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183870&req=5

Figure 4: Multilocus allele analysis of Brucella species and biovar type strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella species and biovars. The allele number reflects the total number of repeats calculated for each locus. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. The star next to the VNTR Locus-4 allele in some samples shows a match with the alternative allele ladder indicating an 11-bp insertion in the non-repeat region of the amplicon.
Mentions: A panel of 19 pure Brucella cultures, composed of all of the Brucella species and biovar FAO/WHO Reference Strains, was assayed by the HOOF-Print procedure. The amplicons obtained from each locus were sized and the numbers of repeat units were determined. The data were compiled in chart format for easy comparison (Fig. 4). The collective alleles amplified from a given isolate formed its genetic fingerprint. Each fingerprint was unique and each of the type strains could be easily differentiated. However, parts of the fingerprint patterns were similar among the related biovars of a single species. The VNTR Locus-8 locus, for example, contained two repeats in all B. abortus biovars. Similarly, the VNTR Locus-3 locus contained a single repeat in the three B. melitensis biovars.

Bottom Line: The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk.The method is rapid and the results are reproducible.HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

Background: Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.

Results: An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.

Conclusion: This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

Show MeSH
Related in: MedlinePlus